RESUMEN
Atrial Fibrillation is the most common sustained cardiac arrhythmia worldwide harming millions of people every year. Atrial Fibrillation (AF) abruptly induces rapid conduction between atrial myocytes which is associated with oxidative stress and abnormal calcium handling. Unfortunately this new equilibrium promotes perpetuation of the arrhythmia. Recently, in addition to being the major source of oxidative stress within cells, mitochondria have been observed to fuse, forming mitochondrial networks and attach to intracellular calcium stores in response to cellular stress. We sought to identify a potential role for rapid stimulation, oxidative stress and mitochondrial hyperfusion in acute changes to myocyte calcium handling. In addition we hoped to link altered calcium handling to increased sarcoplasmic reticulum (SR)-mitochondrial contacts, the so-called mitochondrial associated membrane (MAM). We selected the C2C12 murine myotube model as it has previously been successfully used to investigate mitochondrial dynamics and has a myofibrillar system similar to atrial myocytes. We observed that rapid stimulation of C2C12 cells resulted in mitochondrial hyperfusion and increased mitochondrial colocalisation with calcium stores. Inhibition of mitochondrial fission by transfection of mutant DRP1K38E resulted in similar effects on mitochondrial fusion, SR colocalisation and altered calcium handling. Interestingly the effects of 'forced fusion' were reversed by co-incubation with the reducing agent N-Acetyl cysteine (NAC). Subsequently we demonstrated that oxidative stress resulted in similar reversible increases in mitochondrial fusion, SR-colocalisation and altered calcium handling. Finally, we believe we have identified that myocyte calcium handling is reliant on baseline levels of reactive oxygen species as co-incubation with NAC both reversed and retarded myocyte response to caffeine induced calcium release and re-uptake. Based on these results we conclude that the coordinate regulation of mitochondrial fusion and MAM contacts may form a point source for stress-induced arrhythmogenesis. We believe that the MAM merits further investigation as a therapeutic target in AF-induced remodelling.
Asunto(s)
Calcio/metabolismo , Mitocondrias/metabolismo , Modelos Biológicos , Fibras Musculares Esqueléticas/metabolismo , Estrés Oxidativo , Acetilcisteína/farmacología , Animales , Señalización del Calcio/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/ultraestructura , Dinámicas Mitocondriales/efectos de los fármacos , Membranas Mitocondriales/efectos de los fármacos , Membranas Mitocondriales/metabolismo , Contracción Muscular/efectos de los fármacos , Fibras Musculares Esqueléticas/citología , Fibras Musculares Esqueléticas/ultraestructura , Proteínas Mutantes/metabolismo , Oxidación-Reducción/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Retículo Sarcoplasmático/efectos de los fármacos , Retículo Sarcoplasmático/metabolismoRESUMEN
Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays an important role in cholesterol homeostasis, mediating degradation of the liver low-density lipoprotein receptor (LDLR). In fact, gain- and loss-of-function PCSK9 variations in human populations associate with hyper- or hypo- cholesterolemia, respectively. Exactly how PCSK9 promotes degradation of the LDLR, the identity of the other biomolecules involved in this process, and the global effect of PCSK9 on other proteins has not been thoroughly studied. Here we employ stable isotope labeling with amino acids in cell culture (SILAC) to present the first quantitative, subcellular proteomic study of proteins affected by the stable overexpression of a gain-of-function PCSK9 membrane-bound chimera (PCSK9-V5-ACE2) in comparison to control, empty vector transfections in a human hepatocyte (HuH7) cell line. The expression level of 327 of 5790 peptides was modified by PCSK9-V5-ACE2 overexpression. Immunoblotting was carried out for the control transferrin receptor, shown to be unaffected in cells overexpressing PCSK9-V5-ACE2, thus validating our SILAC results. We also used immunoblotting to confirm the novel SILAC results of up- and down-regulation of several proteins in cells overexpressing PCSK9-V5-ACE2. Moreover, we documented the novel down-regulation of the EH domain binding protein-1 (EHBP1) in a transgenic PCSK9 mouse model and its up-regulation in a PCSK9 knockout mouse model.
Asunto(s)
Hígado/citología , Proteoma/análisis , Proteómica/métodos , Proteínas Recombinantes de Fusión/metabolismo , Serina Endopeptidasas/química , Serina Endopeptidasas/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Línea Celular , Cromatografía Líquida de Alta Presión/métodos , Análisis por Conglomerados , Bases de Datos de Proteínas , Regulación hacia Abajo , Humanos , Marcaje Isotópico/métodos , Ratones , Ratones Noqueados , Ratones Transgénicos , Datos de Secuencia Molecular , Péptidos/análisis , Péptidos/genética , Proproteína Convertasa 9 , Proproteína Convertasas , Receptores de LDL/genética , Receptores de LDL/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Serina Endopeptidasas/genética , Espectrometría de Masas en Tándem/métodos , Regulación hacia ArribaRESUMEN
In this work we report the development of a novel methodology for the determination of stereospecificity of diacyl glycerophospholipids, including glycerophosphatidic acids (PA), glycerophosphoserines (PS), glycerophosphoglycerols (PG), glycerophosphoinositols (PI), and glycerophosphoethanolamines (PE), which can be conventionally ionized in negative ion mode. This methodology uses MS(2) recorded on a hybrid quadrupole time-of-flight mass spectrometer to determine the stereospecificity of diacyl glycerophospholipids based on the lyso-form fragment ions, attributed to the neutral loss of fatty acyl moieties. The fragmentation patterns of a variety of diacyl glycerophospholipid standards were first fully examined over a wide range of collision energy. We observed that lyso-form fragment ions corresponding to the neutral loss of fatty acyl moieties attached to the sn2 position as free fatty acids ([M-Sn2](-) ) and as ketenes ([M-(Sn2-H(2) O)](-) ) exhibited consistently higher intensity than their counterpart ions due to the neutral loss of fatty acyl moieties attached to the sn1 position ([M-Sn1](-) and [M-(Sn1-H(2) O)](-) ). Therefore, we concluded that an empirical fragmentation rule can be used to precisely determine the stereospecificity of diacyl glycerophospholipids, primarily on the basis of relative abundance of the lyso-form fragment ions. We then examined the product ion spectra of diacyl glycerophospholipids recorded from lipid extracts of rat hepatoma cells, where the stereospecific information of these lipids was conclusively determined. Combining the novel methodology reported in this work with the currently widely practiced mass spectrometric techniques such as multiple precursor ion scans (MPIS), fatty acyl scans (FAS), and multidimensional mass spectrometry based shotgun lipidomics (MDMS-SL), should enable a reliable and convenient platform for comprehensive glycerophospholipid profiling.
Asunto(s)
Diglicéridos/química , Glicerofosfolípidos/química , Espectrometría de Masas/métodos , Animales , Lípidos/química , Proteínas Nucleares/metabolismo , Ratas , Estereoisomerismo , Células Tumorales CultivadasRESUMEN
Lipid mediators participate in signal transduction pathways, proliferation, apoptosis, and membrane trafficking in the cell. Lipids are highly complex and diverse owing to the various combinations of polar headgroups, fatty acyl chains, and backbone structures. This structural diversity continues to pose a challenge for lipid analysis. Here we review the current state of the art in lipidomics research and discuss the challenges facing this field. The latest technological developments in mass spectrometry, the role of bioinformatics, and the applications of lipidomics in lipid metabolism and cellular physiology and pathology are also discussed.
Asunto(s)
Metabolismo de los Lípidos/fisiología , Lípidos/química , Metabolómica/métodos , Membrana Celular/ultraestructura , Fenómenos Fisiológicos Celulares , Biología Computacional , Grasas Insaturadas/química , Homeostasis , Humanos , Hidrocarburos/química , Metabolismo de los Lípidos/genética , Lípidos/fisiología , Espectrometría de Masas/instrumentación , Conformación Molecular , Transducción de Señal , Espectrometría de Masas en TándemRESUMEN
Growing evidence links the three mammalian lipin proteins, i.e., lipin-1, lipin-2 and lipin-3, to metabolic and cardiovascular diseases such as noninsulin-dependent diabetes mellitus and atherosclerosis. Lipin proteins play a dual function in lipid metabolism by acting as phosphatidate phosphatase (PAP) enzymes and as transcriptional regulators. Genetic variants within the human LPIN1 and LPIN2 genes are associated with metabolic syndromes. The fatty liver dystrophy (fld) mice carrying mutations within the Lpin1 gene display life-long deficiency in adipogenesis, insulin resistance, neonatal hepatosteatosis and hypertriglyceridemia, as well as increased atherosclerosis susceptibility. Cell culture studies show that hepatic lipin-1 expression is selectively stimulated by glucocorticoids and repressed by insulin, and its subcellular localization governs the assembly and secretion of very low density lipoproteins (VLDL). In noninsulin-dependent diabetes, glucocorticoid signals lead to dyslipidemia characterized by overproduction of VLDL and atherogenic remnants. This puts lipin-1 as a key integrator of hormonal signals to the liver in diabetic dyslipidemia. This review summarizes the current understanding of the role that hepatic lipin-1 plays in the synthesis, storage and compartmentalization of glycerolipids, and highlights the lipid metabolic consequences associated with dysregulated lipin expression.
Asunto(s)
Lípidos/biosíntesis , Lipoproteínas/metabolismo , Hígado/metabolismo , Proteínas Nucleares/metabolismo , Animales , Ratones , Fosfatidato FosfatasaRESUMEN
We have shown that expression of apolipoprotein (apo) C-III promotes VLDL secretion from transfected McA-RH7777 cells under lipid-rich conditions. To determine structural elements within apoC-III that confer to this function, we contrasted wild-type apoC-III with a mutant Ala23Thr originally identified in hypotriglyceridemia subjects. Although synthesis of [(3)H]glycerol-labeled TAG was comparable between cells expressing wild-type apoC-III (C3wt cells) or Ala23Thr mutant (C3AT cells), secretion of [(3)H]TAG from C3AT cells was markedly decreased. The lowered [(3)H]TAG secretion was associated with an inability of C3AT cells to assemble VLDL(1). Moreover, [(3)H]TAG within the microsomal lumen in C3AT cells was 60% higher than that in C3wt cells, yet the activity of microsomal triglyceride-transfer protein in C3AT cells was not elevated. The accumulated [(3)H]TAG in C3AT microsomal lumen was mainly associated with lumenal IDL/LDL-like lipoproteins. Phenotypically, this [(3)H]TAG fractionation profiling resembled what was observed in cells treated with brefeldin A, which at low dose specifically blocked the second-step VLDL(1) maturation. Furthermore, lumenal [(35)S]Ala23Thr protein accumulated in IDL/LDL fractions and was absent in VLDL fractions in C3AT cells. These results suggest that the presence of Ala23Thr protein in lumenal IDL/LDL particles might prevent effective fusion between lipid droplets and VLDL precursors. Thus, the current study reveals an important structural element residing within the N-terminal region of apoC-III that governs the second step VLDL(1) maturation.
Asunto(s)
Apolipoproteínas C/genética , Apolipoproteínas C/metabolismo , Trastornos del Metabolismo de los Lípidos/genética , Mutación Missense , Triglicéridos/metabolismo , Alanina/genética , Apolipoproteínas C/química , Brefeldino A/farmacología , Proteínas Portadoras/metabolismo , Línea Celular , Regulación de la Expresión Génica , Humanos , Lipoproteínas IDL/metabolismo , Lipoproteínas LDL/metabolismo , Lipoproteínas VLDL/metabolismo , Microsomas/metabolismo , Modelos Moleculares , Unión Proteica/efectos de los fármacos , Estructura Secundaria de Proteína , Treonina/genéticaRESUMEN
Apolipoprotein (apo) C-III plays a regulatory role in VLDL lipolysis and clearance. In this study, we determined a potential intracellular role of apoC-III in hepatic VLDL assembly and secretion. Stable expression of recombinant apoC-III in McA-RH7777 cells resulted in increased secretion efficiency of VLDL-associated triacylglycerol (TAG) and apoB-100 in a gene-dosage-dependent manner. The stimulatory effect of apoC-III on TAG secretion was manifested only when cells were cultured under lipid-rich (i.e., media supplemented with exogenous oleate) but not lipid-poor conditions. The stimulated TAG secretion was accompanied by increased secretion of apoB-100 and apoB-48 as VLDL(1). Expression of apoC-III also increased mRNA and activity of microsomal triglyceride transfer protein (MTP). Pulse-chase experiments showed that apoC-III expression promoted VLDL(1) secretion even under conditions where the MTP activity was inhibited immediately after the formation of lipid-poor apoB-100 particles, suggesting an involvement of apoC-III in the second-step VLDL assembly process. Consistent with this notion, the newly synthesized apoC-III was predominantly associated with TAG within the microsomal lumen that resembled lipid precursors of VLDL. Introducing an Ala23-to-Thr mutation into apoC-III, a naturally occurring mutation originally identified in two Mayan Indian subjects with hypotriglyceridemia, abolished the ability of apoC-III to stimulate VLDL secretion from transfected cells. Thus, expression of apoC-III in McA-RH7777 cells enhances hepatic TAG-rich VLDL assembly and secretion under lipid-rich conditions.
Asunto(s)
Apolipoproteína B-100/metabolismo , Apolipoproteína B-48/metabolismo , Apolipoproteína C-III/biosíntesis , Proteínas Portadoras/metabolismo , VLDL-Colesterol/metabolismo , Triglicéridos/metabolismo , Animales , Apolipoproteína C-III/genética , Línea Celular Tumoral , Dosificación de Gen , Técnicas de Silenciamiento del Gen , Humanos , ARN Interferente Pequeño/metabolismo , Ratas , TransfecciónRESUMEN
Phosphatidate phosphatase-1 (PAP-1) converts phosphatidate to diacylglycerol and plays a key role in the biosynthesis of phospholipids and triacylglycerol (TAG). PAP-1 activity is encoded by members of the lipin family, including lipin-1 (1alpha and 1beta), -2, and -3. We determined the effect of lipin-1 expression on the assembly and secretion of very low density lipoproteins (VLDL) using McA-RH7777 cells. Expression of lipin-1alpha or -1beta increased the synthesis and secretion of [(3)H]glycerol-labeled lipids under either basal- or oleate-supplemented conditions. In the presence of oleate, the increased TAG secretion was mainly associated with VLDL(1) (S(f) > 100) and VLDL(2) (S(f) 20-100). Expression of lipin-1alpha or -1beta increased secretion efficiency and decreased intracellular degradation of [(35)S]apolipoprotein B-100 (apoB100). Knockdown of lipin-1 using specific short interfering RNA decreased secretion of [(3)H]glycerolipids and [(35)S]apoB100 even though total PAP-1 activity was not decreased, owing to the presence of lipin-2 and -3 in the cells. Deletion of the nuclear localization signal sequences within lipin-1alpha not only abolished nuclear localization but also resulted in impaired association with microsomal membranes. Cells expressing the cytosolic lipin-1alpha mutant failed to promote [(35)S]apoB100 synthesis or secretion, and showed compromised stimulation in [(3)H]TAG synthesis and secretion. Thus, alteration in hepatic expression of lipin-1 and its compartmentalization control VLDL assembly/secretion.
Asunto(s)
Lipoproteínas VLDL/metabolismo , Hígado/metabolismo , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/genética , Animales , Apolipoproteína B-100/metabolismo , Transporte Biológico , Eliminación de Gen , Regulación de la Expresión Génica , Glicerol/metabolismo , Humanos , Lípidos/química , Ratones , Ratones Noqueados , Microscopía Fluorescente/métodos , Modelos Biológicos , Proteínas Asociadas a Pancreatitis , Fosfatidato FosfatasaRESUMEN
Glucocorticoids (GCs) increase hepatic phosphatidate phosphatase (PAP1) activity. This is important in enhancing the liver's capacity for storing fatty acids as triacylglycerols (TAGs) that can be used subsequently for beta-oxidation or VLDL secretion. PAP1 catalyzes the conversion of phosphatidate to diacylglycerol, a key substrate for TAG and phospholipid biosynthesis. PAP1 enzymes in liver include lipin-1A and -1B (alternatively spliced isoforms) and two distinct gene products, lipin-2 and lipin-3. We determined the mechanisms by which the composite PAP1 activity is regulated using rat and mouse hepatocytes. Levels of lipin-1A and -1B mRNA were increased by dexamethasone (dex; a synthetic GC), and this resulted in increased lipin-1 synthesis, protein levels, and PAP1 activity. The stimulatory effect of dex on lipin-1 expression was enhanced by glucagon or cAMP and antagonized by insulin. Lipin-2 and lipin-3 mRNA were not increased by dex/cAMP, indicating that increased PAP1 activity is attributable specifically to enhanced lipin-1 expression. This work provides the first evidence for the differential regulation of lipin activities. Selective lipin-1 expression explains the GC and cAMP effects on increased hepatic PAP1 activity, which occurs in hepatic steatosis during starvation, diabetes, stress, and ethanol consumption.
Asunto(s)
AMP Cíclico/farmacología , Glucocorticoides/farmacología , Hígado/metabolismo , Proteínas Nucleares/genética , Fosfotransferasas (Aceptor del Grupo Fosfato)/metabolismo , Animales , Técnicas de Cultivo de Célula , Ingestión de Alimentos , Ayuno , Regulación de la Expresión Génica/efectos de los fármacos , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Hígado/efectos de los fármacos , Masculino , Proteínas Asociadas a Pancreatitis , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética/efectos de los fármacosRESUMEN
Sperm-zona pellucida (ZP) binding is the first step of gamete interaction. This binding occurs in two sequential steps, starting with the primary binding of acrosome-intact sperm to the ZP followed by the secondary ZP binding of acrosome reacting/reacted sperm. While there are only a few ZP sulfoglycoproteins involved in these binding events, a large number of sperm surface molecules have been shown to possess ZP affinity. In this review, we have given explanations to the existence of these many ZP binding molecules. We have also summarized their origin and the mechanisms of how they are targeted to the sperm surface and acrosome. Recently, we have shown that sperm lipid rafts have affinity for the ZP. A number of ZP binding molecules are also present in sperm lipid rafts. In this review, we have provided an argument that sperm lipid rafts may be the platforms on the sperm surface for ZP interaction.
Asunto(s)
Microdominios de Membrana/metabolismo , Interacciones Espermatozoide-Óvulo , Espermatozoides/química , Zona Pelúcida/metabolismo , Animales , Sitios de Unión , Humanos , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Cabeza del Espermatozoide/metabolismo , Espermatozoides/metabolismoRESUMEN
Sperm gain full ability to bind to the zona(e) pellucida(e) (ZP) during capacitation. Since lipid rafts are implicated in cell adhesion, we determined whether capacitated sperm lipid rafts had affinity for the ZP. We demonstrated that lipid rafts, isolated as low-density detergent resistant membranes (DRMs), from capacitated pig sperm had ability to bind to homologous ZP. This binding was dependent on pig ZPB glycoprotein, a major participant in sperm binding. Capacitated sperm DRMs were also enriched in the male germ cell specific sulfogalactosylglycerolipid (SGG), which contributed to DRMs-ZP binding. Furthermore, SGG may participate in the formation of sperm DRMs due to its interaction with cholesterol, an integral component of lipid rafts, as shown by infrared spectroscopic studies. Since sperm capacitation is associated with cholesterol efflux from the sperm membrane, we questioned whether the formation of DRMs was compromised in capacitated sperm. Our studies indeed revealed that capacitation induced increased levels of sperm DRMs, with an enhanced ZP affinity. These results corroborated the implication of lipid rafts and SGG in cell adhesion and strongly suggested that the enhanced ZP binding ability of capacitated sperm may be attributed to increased levels and a greater ZP affinity of lipid rafts in the sperm plasma membrane.
Asunto(s)
Colesterol/metabolismo , Galactolípidos/fisiología , Microdominios de Membrana/metabolismo , Capacitación Espermática/fisiología , Zona Pelúcida/fisiología , Animales , Membrana Celular/metabolismo , Femenino , Fertilización/fisiología , Masculino , Lípidos de la Membrana/metabolismo , Fosforilación , Cabeza del Espermatozoide/fisiología , PorcinosRESUMEN
Although Percoll gradient centrifugation has been used routinely to prepare motile human sperm, its use in preparing motile mouse sperm has been limited. Here, we showed that Percoll gradient-centrifuged (PGC) capacitated mouse sperm had markedly higher fertilizing ability (sperm-zona pellucida [ZP] binding and in vitro fertilization) than washed capacitated mouse sperm. We also showed that the lipid profiles of PGC capacitated sperm and washed capacitated sperm differed significantly. The PGC sperm had much lower contents of cholesterol and phospholipids. This resulted in relative enrichment of male germ cell-specific sulfogalactosylglycerolipid (SGG), a ZP-binding ligand, in PGC capacitated sperm, and this would explain, in part, their increased ZP-binding ability compared with that of washed capacitated sperm. Analyses of phospholipid fatty acyl chains revealed that PGC capacitated sperm were enriched in phosphatidylcholine (PC) molecular species containing highly unsaturated fatty acids (HUFAs), with docosahexaenoic acid (DHA; C22: 6n-3) being the predominant HUFA (42% of total hydrocarbon chains of PC). In contrast, the level of PC-HUFAs comprising arachidonic acid (20:4n-6), docosapentaenoic acid (C22:5n-6), and DHA in washed capacitated sperm was only 27%. Having the highest unsaturation degree among all HUFAs in PC, DHA would enhance membrane fluidity to the uppermost. Therefore, membranes of PGC capacitated sperm would undergo fertilization-related fusion events at higher rates than washed capacitated sperm. These results suggested that PGC mouse sperm should be used in fertilization experiments and that SGG and DHA should be considered to be important biomarkers for sperm fertilizing ability.